Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals.

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.

Inhibition of Glycogen Synthase II with RNAi Prevents Liver Injury in Mouse Models of Glycogen Storage Diseases.

Glycogen storage diseases (GSDs) of the liver are devastating disorders presenting with fasting hypoglycemia as well as hepatic glycogen and lipid accumulation, which could lead to long-term liver damage. Diet control is frequently utilized to manage the potentially dangerous hypoglycemia, but there is currently no effective pharmacological treatment for preventing hepatomegaly and concurrent liver metabolic abnormalities, which could lead to fibrosis, cirrhosis, and hepatocellular adenoma or carcinoma. In this study, we demonstrate that inhibition of glycogen synthesis using an RNAi approach to silence hepatic Gys2 expression effectively prevents glycogen synthesis, glycogen accumulation, hepatomegaly, fibrosis, and nodule development in a mouse model of GSD III. Mechanistically, reduction of accumulated abnormally structured glycogen prevents proliferation of hepatocytes and activation of myofibroblasts as well as infiltration of mononuclear cells. Additionally, we show that silencing Gys2 expression reduces hepatic steatosis in a mouse model of GSD type Ia, where we hypothesize that the reduction of glycogen also reduces the production of excess glucose-6-phosphate and its subsequent diversion to lipid synthesis. Our results support therapeutic silencing of GYS2 expression to prevent glycogen and lipid accumulation, which mediate initial signals that subsequently trigger cascades of long-term liver injury in GSDs.

Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants.

Since the establishment of a canonical animal microRNA biogenesis pathway driven by the RNase III enzymes Drosha and Dicer, an unexpected variety of alternative mechanisms that generate functional microRNAs have emerged. We review here the many Drosha-independent and Dicer-independent microRNA biogenesis strategies characterized over the past few years. Beyond reflecting the flexibility of small RNA machineries, the existence of noncanonical pathways has consequences for interpreting mutants in the core microRNA machinery. Such mutants are commonly used to assess the consequences of "total" microRNA loss, and indeed, they exhibit many overall phenotypic similarities. Nevertheless, ongoing studies reveal a growing number of settings in which alternative microRNA pathways contribute to distinct phenotypes among core microRNA biogenesis mutants.

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila.

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼ 23-26 nt products. The difference was due to the 3' modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3' trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3'-5' exoribonuclease that trims 'long' mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.

A genome-wide transgenic resource for conditional expression of Drosophila microRNAs.

microRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.

The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and Drosophila.

MicroRNA (miRNA) biogenesis proceeds from a primary transcript (pri-miRNA) through the pre-miRNA into the mature miRNA. Here, we identify a role of the Caenorhabditis elegans nuclear export receptor XPO-1 and the cap-binding proteins CBP-20/NCBP-2 and CBP-80/NCBP-1 in this process. The RNA-mediated interference of any of these genes causes retarded heterochronic phenotypes similar to those observed for animals with mutations in the let-7 miRNA or core miRNA machinery genes. Moreover, pre- and mature miRNAs become depleted, whereas primary miRNA transcripts accumulate. An involvement of XPO-1 in miRNA biogenesis is conserved in Drosophila, in which knockdown of Embargoed/XPO-1 or its chemical inhibition through leptomycin B causes pri-miRNA accumulation. Our findings demonstrate that XPO-1/Emb promotes the pri-miRNA-to-pre-miRNA processing and we propose that this function involves intranuclear transport and/or nuclear export of primary miRNAs.

Functional parameters of Dicer-independent microRNA biogenesis.

Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.

Widespread regulatory activity of vertebrate microRNA* species.

An obligate intermediate during microRNA (miRNA) biogenesis is an ~22-nucleotide RNA duplex, from which the mature miRNA is preferentially incorporated into a silencing complex. Its partner miRNA* species is generally regarded as a passenger RNA, whose regulatory capacity has not been systematically examined in vertebrates. Our bioinformatic analyses demonstrate that a substantial fraction of miRNA* species are stringently conserved over vertebrate evolution, collectively exhibit greatest conservation in their seed regions, and define complementary motifs whose conservation across vertebrate 3'-UTR evolution is statistically significant. Functional tests of 22 miRNA expression constructs revealed that a majority could repress both miRNA and miRNA* perfect match reporters, and the ratio of miRNA:miRNA* sensor repression was correlated with the endogenous ratio of miRNA:miRNA* reads. Analysis of microarray data provided transcriptome-wide evidence for the regulation of seed-matched targets for both mature and star strand species of several miRNAs relevant to oncogenesis, including mir-17, mir-34a, and mir-19. Finally, 3'-UTR sensor assays and mutagenesis tests confirmed direct repression of five miR-19* targets via star seed sites. Overall, our data demonstrate that miRNA* species have demonstrable impact on vertebrate regulatory networks and should be taken into account in studies of miRNA functions and their contribution to disease states.

A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs.

Canonical animal microRNAs (miRNAs) are ∼22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. We performed a genetic screen using an miRNA-repressed reporter in the Drosophila eye and recovered the first reported alleles of fly drosha, an allelic series of its dsRBD partner pasha, and novel alleles of dicer-1. Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. We further used these mutants to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, we unexpectedly recovered hypomorphic alleles that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system.

Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis.

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3' terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.

Homeostatic control of Argonaute stability by microRNA availability.

Homeostatic mechanisms regulate the abundance of several components in small-RNA pathways. We used Drosophila and mammalian systems to demonstrate a conserved homeostatic system in which the status of miRNA biogenesis controls Argonaute protein stability. Clonal analyses of multiple mutants of core Drosophila miRNA factors revealed that stability of the miRNA effector AGO1 is dependent on miRNA biogenesis. Reciprocally, ectopic transcription of miRNAs within in vivo clones induced accumulation of AGO1, as did genetic interference with the ubiquitin-proteasome system. In mouse cells, we found that the stability of Ago2 declined in Dicer-knockout cells and was rescued by proteasome blockade or introduction of either Dicer plasmid or Dicer-independent miRNA constructs. Notably, Dicer-dependent miRNA constructs generated pre-miRNAs that bound Ago2 but did not rescue Ago2 stability. We conclude that Argonaute levels are finely tuned by cellular availability of mature miRNAs and the ubiquitin-proteasome system.

Dicer-independent, Ago2-mediated microRNA biogenesis in vertebrates.

A canonical biogenesis pathway involving sequential cleavage by the Drosha and Dicer RNAse III enzymes governs the maturation of most animal microRNAs. However, there exist a variety of alternative miRNA biogenesis pathways, most of which bypass Drosha processing. Recently, three groups described for the first time a vertebrate microRNA pathway that bypasses Dicer cleavage. This mechanism was characterized with respect to the highly conserved vertebrate gene mir-451, for which Drosha processing yields a short (42 nucleotide) hairpin that is directly loaded into Ago2, the sole vertebrate "Slicer" Argonaute. Ago2-mediated cleavage of this hairpin yields a 30 nucleotide intermediate, whose 3' end is resected to generate the dominantly cloned ~23 nucleotide mature miR-451. Knowledge of this pathway provides an unprecedented tool with which to express microRNAs and small interfering RNAs in Dicer mutant cells. More generally, the mir-451 backbone constitutes a new platform for gene silencing that complements existing shRNA technology.

The Drosophila miR-310 cluster negatively regulates synaptic strength at the neuromuscular junction.

Emerging data implicate microRNAs (miRNAs) in the regulation of synaptic structure and function, but we know little about their role in the regulation of neurotransmission in presynaptic neurons. Here, we demonstrate that the miR-310-313 cluster is required for normal synaptic transmission at the Drosophila larval neuromuscular junction. Loss of miR-310-313 cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of mir-310-313 in larval presynaptic neurons. Kinesin family member, Khc-73 is a functional target for miR-310-313 as its expression is increased in mir-310-313 mutants and reducing it restores normal synaptic function. Cluster mutants show an increase in the active zone protein Bruchpilot accompanied by an increase in electron dense T bars. Finally, we show that repression of Khc-73 by miR-310-313 cluster influences the establishment of normal synaptic homeostasis. Our findings establish a role for miRNAs in the regulation of neurotransmitter release.